Indole metabolites generated by microbiota inhibit Cryptosporidium growth

Funkhouser-Jones et al. recently identified gut metabolites that affected Cryptosporidium growth. A key focus, indole, was shown to inhibit the parasite in vivo and in vitro by decreasing the host mitochondria function and the membrane potential of parasite mitosomes. These findings help clarify the role microflora and metabolites play in host resistance.     

Fomiroid A,a Novel Compound from the Mushroom Fomitopsis nigra,Inhibits NPC1L1-Mediated Cholesterol Uptake via a Mode of Action Distinct from That of Ezetimibe

Hypercholesterolemia is one of the key risk factors for coronary heart disease, a major cause of death in developed countries. Suppression of NPC1L1-mediated dietary and biliary cholesterol absorption is predicted to be one of the most effective ways to reduce the risk of hypercholesterolemia. In a screen for natural products that inhibit ezetimibe glucuronide binding to NPC1L1, we found a novel compound, fomiroid A, in extracts of the mushroom Fomitopsis nigra. Fomiroid A is a lanosterone derivative with molecular formula C₃₀H₄₈O₃. Fomiroid A inhibited ezetimibe glucuronide binding to NPC1L1, and dose-dependently prevented NPC1L1-mediated cholesterol uptake and formation of esterified cholesterol in NPC1L1-expressing Caco2 cells. Fomiroid A exhibited a pharmacological chaperone activity that corrected trafficking defects of the L1072T/L1168I mutant of NPC1L1. Because ezetimibe does not have such an activity, the binding site and mode of action of fomiroid A are likely to be distinct from those of ezetimibe.     

The molecular mechanisms underlying the reduction of LDL apoB-100 by ezetimibe plus simvastatin

The combination of ezetimibe, an inhibitor of Niemann-Pick C1-like 1 protein (NPC1L1), and an HMG-CoA reductase inhibitor decreases cholesterol absorption and synthesis. In clinical trials, ezetimibe plus simvastatin produces greater LDL-cholesterol reductions than does monotherapy. The molecular mechanism for this enhanced efficacy has not been defined. Apolipoprotein B-100 (apoB-100) kinetics were determined in miniature pigs treated with ezetimibe (0.1 mg/kg/day), ezetimibe plus simvastatin (10 mg/kg/day), or placebo (n = 7/group). Ezetimibe decreased cholesterol absorption (-79%) and plasma phytosterols (-91%), which were not affected further by simvastatin. Ezetimibe increased plasma lathosterol (+65%), which was prevented by addition of simvastatin. The combination decreased total cholesterol (-35%) and LDL-cholesterol (-47%). VLDL apoB pool size decreased 26%, due to a 35% decrease in VLDL apoB production. LDL apoB pool size decreased 34% due to an 81% increase in the fractional catabolic rate, both of which were significantly greater than monotherapy. Combination treatment decreased hepatic microsomal cholesterol (-29%) and cholesteryl ester (-65%) and increased LDL receptor (LDLR) expression by 240%. The combination increased NPC1L1 expression in liver and intestine, consistent with increased SREBP2 expression. Ezetimibe plus simvastatin decreases VLDL and LDL apoB-100 concentrations through reduced VLDL production and upregulation of LDLR-mediated LDL clearance.     

Niemann–Pick C1 Like 1 (NPC1L1) an intestinal sterol transporter

Niemann–Pick C1 Like 1 (NPC1L1) has been identified and characterized as an essential protein in the intestinal cholesterol absorption process. NPC1L1 localizes to the brush border membrane of absorptive enterocytes in the small intestine. Intestinal expression of NPC1L1 is down regulated by diets containing high levels of cholesterol. While otherwise phenotypically normal, Npc1l1 null mice exhibit a significant reduction in the intestinal uptake and absorption of cholesterol and phytosterols. Characterization of the NPC1L1 pathway revealed that cholesterol absorption inhibitor ezetimibe specifically binds to an extracellular loop of NPC1L1 and inhibits its sterol transport function. Npc1l1 null mice are resistant to diet-induced hypercholesterolemia, and when crossed with apo E null mice, are completely resistant to the development of atherosclerosis. Intestinal gene expression studies in Npc1l1 null mice indicated that no exogenous cholesterol was entering enterocytes lacking NPC1L1, which resulted in an upregulation of intestinal and hepatic LDL receptor and cholesterol biosynthetic gene expression. Polymorphisms in the human NPC1L1 gene have been found to influence cholesterol absorption and plasma low density lipoprotein levels. Therefore, NPC1L1 is a critical intestinal sterol uptake transporter which influences whole body cholesterol homeostasis.     

Cryptosporidium parvum scavenges LDL‐derived cholesterol and micellar cholesterol internalized into enterocytes

Cryptosporidium spp. are responsible for devastating diarrhoea in immunodeficient individuals. In the intestinal tract, the developmental stages of the parasite are confined to the apical surfaces of epithelial cells. Upon invasion, Cryptosporidium incorporates the microvillous membrane of the enterocyte to form the parasitophorous vacuole (PV) and sequesters itself from the host cytoplasm by rearranging the host cytoskeleton. Cryptosporidium parvum has minimal anabolic capabilities and relies on transporters and salvage pathways to meet its basic metabolic requirements. The cholesterol salvage pathway is crucial for the development of protozoan parasites. In this study, we have examined the sources of cholesterol from C. parvum infecting enterocytes. We illustrated that the intracellular stages of Cryptosporidium as well as the oocysts shed by the host, contain cholesterol. Incubation of infected enterocytes in lipoprotein‐free medium impairs parasite development and results in substantial decrease in cholesterol content associated with the PV. Among lipoproteins, LDL constitutes an important source of cholesterol for Cryptosporidium. Dietary cholesterol incorporated into micelles is internalized into enterocytes by the NPC1L1 transporter. We showed that C. parvum also obtains cholesterol from micelles in enterocytes.Pharmacological blockade of NPC1L1 function by ezetimibe or moderate downregulation of NPC1L1 expression decreases parasite infectivity. These observations indicate that, despite its dual sequestration from the intestinal lumen and the host cytoplasm, C. parvum can, in fact, obtain cholesterol both from the gut's lumen and the host cell. This study highlights the evolutionary advantages for epicellular pathogens to access to nutrients from the outside and inside of the host cell.     

Luteolin and Quercetin Affect the Cholesterol Absorption Mediated by Epithelial Cholesterol Transporter Niemann–Pick C1-Like 1 in Caco-2 Cells and Rats

Niemann–Pick C1-Like 1 (NPC1L1) mediates cholesterol absorption, and ezetimibe is a potent NPC1L1 inhibitor applicable for medication of hypercholesterolemia. Epidemiological studies demonstrated that consumption of polyphenols correlates with a decreased risk for atherosclerosis due to their antioxidant effect. This activity can hardly be attributable to the antioxidant activity only, and we hypothesized that polyphenols inhibit intestinal transport of cholesterol. We elucidated the kinetic parameters of intestinal cholesterol absorption, screened several polyphenols for their ability to specifically inhibit intestinal cholesterol absorption, and determined the inhibitory effects of selected flavonoids in vitro and in vivo. The concentration-dependent uptake of cholesterol by Caco-2 cells obeyed a monophasic saturation process. This indicates the involvement of an active-passive transport, i.e., NPC1L1. Parameters of cholesterol uptake by Caco-2 cells were as follows: J _max, K _t, and K _d were 6.89±2.96 19.03±11.58 µM, and 0.11±0.02 pmol/min/mg protein, respectively. Luteolin and quercetin inhibited cholesterol absorption by Caco-2 cells and human embryonic kidney 293T cells expressing NPC1L1. When preincubated Caco-2 cells with luteolin and quercetin before the assay, cholesterol uptake significantly decreased. The inhibitory effects of these flavonoids were maintained for up to 120 min. The level of inhibition and irreversible effects were similar to that of ezetimibe. Serum cholesterol levels significantly decreased more in rats fed both cholesterol and luteolin (or quercetin), than in those observed in the cholesterol feeding group. As quercetin induced a significant decrease in the levels of NPC1L1 mRNA in Caco-2 cells, the in vivo inhibitory effect may be due to the expression of NPC1L1. These results suggest that luteolin and quercetin reduce high blood cholesterol levels by specifically inhibiting intestinal cholesterol absorption mediated by NPC1L1.     

Ezetimibe interferes with cholesterol trafficking from the plasma membrane to the endoplasmic reticulum in CaCo-2 cells

Niemann-Pick C1-like 1 protein (NPC1L1) is the putative intestinal sterol transporter and the molecular target of ezetimibe, a potent inhibitor of cholesterol absorption. To address the role of NPC1L1 in cholesterol trafficking in intestine, the regulation of cholesterol trafficking by ezetimibe was studied in the human intestinal cell line, CaCo-2. Ezetimibe caused only a modest decrease in the uptake of micellar cholesterol, but markedly prevented its esterification. Cholesterol trafficking from the plasma membrane to the endoplasmic reticulum was profoundly disrupted by ezetimibe without altering the trafficking of cholesterol from the endoplasmic reticulum to the plasma membrane. Cholesterol oxidase-accessible cholesterol at the apical membrane was increased by ezetimibe. Cholesterol synthesis was modestly increased. Although the amount of cholesteryl esters secreted at the basolateral membrane was markedly decreased by ezetimibe, the transport of lipids and the number of lipoprotein particles secreted were not altered. NPC1L1 gene and protein expression were decreased by sterol influx, whereas cholesterol depletion enhanced NPC1L1 gene and protein expression. These results suggest that NPC1L1 plays a role in cholesterol uptake and cholesterol trafficking from the plasma membrane to the endoplasmic reticulum. Interfering with its function will profoundly decrease the amount of cholesterol transported into lymph.     

Actin subversion for productive Plasmodium hepatocyte invasion

Productive invasion of hepatocytes by Plasmodium sporozoites is a key step of infection. The parasites traverse hepatocytes before targeting one of them to form a parasitophorous vacuole for parasite expansion. Schepis et al. show the induction of membrane ruffling via host Rho GTPases by Plasmodium sporozoites facilitating productive invasion.     

Changes in cholesterol absorption and cholesterol synthesis caused by ezetimibe and/or simvastatin in men

This study evaluates changes in cholesterol balance in hypercholesterolemic subjects following treatment with an inhibitor of cholesterol absorption or cholesterol synthesis or coadministration of both agents. This was a randomized, double blind, placebo-controlled, four-period crossover study to evaluate the effects of coadministering 10 mg ezetimibe with 20 mg simvastatin (ezetimibe/simvastatin) on cholesterol absorption and synthesis relative to either drug alone or placebo in 41 subjects. Each treatment period lasted 7 weeks. Ezetimibe and ezetimibe/simvastatin decreased fractional cholesterol absorption by 65% and 59%, respectively (P < 0.001 for both relative to placebo). Simvastatin did not significantly affect cholesterol absorption. Ezetimibe and ezetimibe/simvastatin increased fecal sterol excretion (corrected for dietary cholesterol), which also represents net steady state cholesterol synthesis, by 109% and 79%, respectively (P < 0.001). Ezetimibe, simvastatin, and ezetimibe/simvastatin decreased plasma LDL-cholesterol by 20, 38, and 55%, respectively. The coadministered therapy was well tolerated. The decreases in net cholesterol synthesis and increased fecal sterol excretion yielded nearly additive reductions in LDL-cholesterol for the coadministration of ezetimibe and simvastatin.     

Human NPC1L1 and NPC1 can functionally substitute for the ncr genes to promote reproductive development in C. elegans

The NPC1 and NPC1L1 are related genes whose general role is in cholesterol trafficking. However, reduction of activity of these genes results in very different phenotypes. Niemann–Pick C disease type 1 is a neurodegenerative disease with no current treatment, where cholesterol accumulates in lysosomes. The disease arises due to autosomal recessive mutations in the NPC1 gene. The NPC1L1 gene has recently been identified as the target for the drug ezetimibe (Zetia), a cholesterol absorption inhibitor, and has been shown to be an intestinal cholesterol transporter. We demonstrate that human NPC1L1, as well as human NPC1, can functionally substitute for the Caenorhabditis elegans genes ncr-1 and/or ncr-2. These genes are known to play a role in the process of dauer formation, a process which can be modulated by cholesterol in sensitized genetic backgrounds. Our results demonstrate that these human proteins retain some functional conservation, though their biological roles are vastly different.     

Regulation of the expression of cholesterol transporters by lipid-lowering drugs ezetimibe and pemafibrate in rat liver and intestine

Ezetimibe and pemafibrate are lipid-lowering drugs and promote reverse cholesterol transport. However, it is unknown whether cholesterol is mainly excreted by hepatobiliary excretion or by non-biliary transintestinal cholesterol efflux (TICE). We evaluated the effects of ezetimibe and pemafibrate on hepatic and intestinal cholesterol transporter regulation in Sham-operated rats, and examined the effects of these drugs on TICE in bile duct-ligated rats. Seven-week-old male Sprague-Dawley rats were treated as follows for two weeks: 1) Sham, Sham operation; 2) BDL, bile duct ligation; 3) E-Sham, Sham + ezetimibe; 4) E-BDL, BDL + ezetimibe; 5) P-Sham, Sham + pemafibrate; and 6) P-BDL, BDL + pemafibrate. Blood, liver, jejunum, and feces were collected 72 h post-surgery. Hepatic cholesterol levels were decreased in P-Sham and E-Sham, and were lower in E-BDL and P-BDL than in BDL. Fecal cholesterol levels increased in E-Sham and P-Sham compared with Sham, and were higher in E-BDL and P-BDL than in BDL. In liver, Abcg5 mRNA showed induction in E-Sham, Abcg5 and Abca1 mRNA were induced in P-Sham, Abcg5 mRNA was reduced in E-BDL, and Abca1 mRNA was increased in P-BDL. In jejunum, Abcg5 mRNA was induced in E-Sham. Abcg8 mRNA was induced in E-Sham and P-Sham. NPC1L1 mRNA showed reduced expression in P-Sham and P-BDL. SR-B1 mRNA was reduced in P-Sham, and the expression decreased in P-BDL. LDL receptor mRNA was induced in BDL and P-BDL. Ezetimibe and pemafibrate may promote TICE by increasing Abcg5/g8, while pemafibrate may inhibit intestinal cholesterol absorption by decreasing SR-B1 and NPC1L1.     

Converting and hoarding driven by protein phosphorylation in Toxoplasma gondii

Successful parasitism relies on the evasion of adversarial host responses. Wang et al. have recently shown that Toxoplasma gondii relies on the protein phosphatase 2A (PP2A) to cause persisting infections. The phosphatase controls the development of dormant parasite stages and the accumulation of sugar supplies.     

Ezetimibe impairs transcellular lipid trafficking and induces large lipid droplet formation in intestinal absorptive epithelial cells

Ezetimibe inhibits Niemann-Pick C1-like 1 (NPC1L1) protein, which mediates intracellular cholesterol trafficking from the brush border membrane to the endoplasmic reticulum, where chylomicron assembly takes place in enterocytes or in the intestinal absorptive epithelial cells. Cholesterol is a minor lipid constituent of chylomicrons; however, whether or not a shortage of cholesterol attenuates chylomicron assembly is unknown. The aim of this study was to examine the effect of ezetimibe, a potent NPC1L1 inhibitor, on trans-epithelial lipid transport, and chylomicron assembly and secretion in enterocytes. Caco-2 cells, an absorptive epithelial model, grown onto culture inserts were given lipid micelles from the apical side, and chylomicron-like triacylglycerol-rich lipoprotein secreted basolaterally were analyzed after a 24-h incubation period in the presence of ezetimibe up to 50 μM. The secretion of lipoprotein and apolipoprotein B48 were reduced by adding ezetimibe (30% and 34%, respectively). Although ezetimibe allowed the cells to take up cholesterol normally, the esterification was abolished. Meanwhile, oleic acid esterification was unaffected. Moreover, ezetimibe activated sterol regulatory element-binding protein 2 by approximately 1.5-fold. These results suggest that ezetimibe limited cellular cholesterol mobilization required for lipoprotein assembly. In such conditions, large lipid droplet formation in Caco-2 cells and the enterocytes of mice were induced, implying that unprocessed triacylglycerol was sheltered in these compartments. Although ezetimibe did not reduce the post-prandial lipid surge appreciably in triolein-infused mice, the results of the present study indicated that pharmacological actions of ezetimibe may participate in a novel regulatory mechanism for the efficient chylomicron assembly and secretion.     

Modulation of lipid metabolism with the overexpression of NPC1L1 in mouse liver

Niemann-Pick C1-like 1 protein (NPC1L1), a transporter crucial in intestinal cholesterol absorption, is expressed in human liver but not in murine liver. To elucidate the role of hepatic NPC1L1 on lipid metabolism, we overexpressed NPC1L1 in murine liver utilizing adenovirus-mediated gene transfer. C57BL/6 mice, fed on normal chow with or without ezetimibe, were injected with NPC1L1 adenovirus (L1-mice) or control virus (Null-mice), and lipid analyses were performed five days after the injection. The plasma cholesterol levels increased in L1-mice, and FPLC analyses revealed increased cholesterol contents in large HDL lipoprotein fractions. These fractions, which showed α-mobility on agarose electrophoresis, were rich in apoE and free cholesterol. These lipoprotein changes were partially inhibited by ezetimibe treatment and were not observed in apoE-deficient mice. In addition, plasma and VLDL triglyceride (TG) levels decreased in L1-mice. The expression of microsomal triglyceride transfer protein (MTP) was markedly decreased in L1-mice, accompanied by the reduced protein levels of forkhead box protein O1 (FoxO1). These changes were not observed in mice with increased hepatic de novo cholesterol synthesis. These data demonstrate that cholesterol absorbed through NPC1L1 plays a distinct role in cellular and plasma lipid metabolism, such as the appearance of apoE-rich lipoproteins and the diminished VLDL-TG secretion.     

Sideways: road to gene-by-gene functional screening in malaria parasites

Genome-wide screening in apicomplexan species has transformed our understanding of these parasitic protozoa. Kimmel et al. report a 'knock sideways' system and provide a powerful use case for its feasibility in a gene-by-gene screening in Plasmodium falciparum. Carefully deployed, a novel toolkit helps to dissect the biological uniqueness of an important parasite.     

Ezetimibe restores biliary cholesterol excretion in mice expressing Niemann-Pick C1-Like 1 only in liver

Niemann-Pick C1-Like 1 (NPC1L1) is highly expressed in the small intestine across mammalian species and is the target of ezetimibe, a potent cholesterol absorption inhibitor. In humans, NPC1L1 is also expressed in the liver. We found that transgenic overexpression of NPC1L1 in the wild-type mouse liver inhibits biliary cholesterol secretion and raises blood cholesterol, which can be reversed by ezetimibe treatment. Unfortunately, the high expression of endogenous NPC1L1 in the intestine hampered a definitive establishment of the role of hepatic NPC1L1 in cholesterol metabolism and ezetimibe action in the liver because intestinal NPC1L1 dramatically influences cholesterol homeostasis and is a target of ezetimibe. To circumvent this obstacle, we crossed liver-specific NPC1L1 transgenic mice to NPC1L1 knockout (L1-KO) mice and created a mouse line expressing no endogenous NPC1L1, but human NPC1L1 in liver only (L1(LivOnly) mice). Compared to L1-KO mice, L1(LivOnly) mice on a 0.2% cholesterol diet showed significantly increased hepatic and plasma cholesterol, and despite a 90% reduction in biliary cholesterol excretion, their fecal cholesterol excretion remained completely unaltered. Remarkably, 4days of ezetimibe treatment significantly restored biliary cholesterol secretion in L1(LivOnly) mice. These findings demonstrated a direct role of hepatic NPC1L1 in regulating biliary cholesterol excretion and hepatic/blood cholesterol levels, and unequivocally established hepatic NPC1L1 as a target of ezetimibe.     

Niemann-Pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole-body cholesterol homeostasis

Niemann-Pick C1 Like 1 (NPC1L1) is a protein localized in jejunal enterocytes that is critical for intestinal cholesterol absorption. The uptake of intestinal phytosterols and cholesterol into absorptive enterocytes in the intestine is not fully defined on a molecular level, and the role of NPC1L1 in maintaining whole body cholesterol homeostasis is not known. NPC1L1 null mice had substantially reduced intestinal uptake of cholesterol and sitosterol, with dramatically reduced plasma phytosterol levels. The NPC1L1 null mice were completely resistant to diet-induced hypercholesterolemia, with plasma lipoprotein and hepatic cholesterol profiles similar to those of wild type mice treated with the cholesterol absorption inhibitor ezetimibe. Cholesterol/cholate feeding resulted in down-regulation of intestinal NPC1L1 mRNA expression in wild type mice. NPC1L1 deficiency resulted in up-regulation of intestinal hydroxymethylglutaryl-CoA synthase mRNA and an increase in intestinal cholesterol synthesis, down-regulation of ABCA1 mRNA, and no change in ABCG5 and ABCG8 mRNA expression. NPC1L1 is required for intestinal uptake of both cholesterol and phytosterols and plays a major role in cholesterol homeostasis. Thus, NPC1L1 may be a useful drug target for the treatment of hypercholesterolemia and sitosterolemia.     

Dual function of the CHS3-CSA1 immune receptor pair

Plant nucleotide-binding leucine-rich repeat (NLR) receptors mediate specific recognition of pathogen effectors to initiate effector-triggered immunity. Recently, studies by Schulze et al., Yang et al., and Gu et al. collectively show that the Arabidopsis (Arabidopsis thaliana) NLR pair CHS3-CSA1 acts through two distinct activation modes to recognize different pathogen effectors, thus revealing the dual function of the CHS3-CSA1 pair in plant disease resistance.     

CONSTANS,a key-player connecting day length to seed size

Plants sense oscillation in the day length as a reliable seasonal cue to drive optimal vegetative and reproductive growth. A recent study by Yu et al. has revealed how day length regulates seed size through CONSTANS. The CONSTANS–APETALA2 module enables plants to optimize their reproductive growth based on their photoperiod response type.     

Feeding the world sustainably: efficient nitrogen use

Globally, overuse of nitrogen (N) fertilizers in croplands is causing severe environmental pollution. In this context, Gu et al. suggest environmentally friendly and cost-effective N management practices and Hamani et al. highlight the use of microbial inoculants to improve crop yields, while reducing N-associated environmental pollution and N-fertilizer use.